Immunoprotein-Mediated siRNA Delivery - ACS Publications

Review pubs.acs.org/molecularpharmaceutics

Immunoprotein-Mediated siRNA Delivery Nicole Baü mer, Wolfgang E. Berdel, and Sebastian Baü mer*

Mol. Pharmaceutics 2017.14:1339-1351. Downloaded from pubs.acs.org by DURHAM UNIV on 10/02/18. For personal use only.

Deparment of Medicine A, Hematology and Oncology, University Hospital Muenster, Albert-Schweitzer Campus 1, Muenster, DE 48149, Germany ABSTRACT: RNA interference strategies offer an alternative to small molecular drug targeting. Small interfering RNA (siRNA) constitutes a class of molecules that allows the effective and specific inhibition of the biosynthesis of any protein. Indeed, siRNA have emerged as a major tool in molecular biology techniques and an important approach to identify suitable therapy targets in cancer. However, siRNA therapy approaches in vivo are scarce. Two major problems hinder siRNA as a therapeutic tool: (1) delivery through the bloodstream leads to degradation or rapid renal clearance (stabilization) and (2) specific uptake by the desired cell type (specificity). This review summarizes the ongoing attempts to use RNAi against disease-causing factors. We compare methods to stabilize siRNA in different conjugates and that decorate these complexes with targeting molecules such as antibodies, single-chain Fv or Fab fragments, to enable specific uptake of the carriers by the respective cells. We propose the development of antibody-coupled siRNA complexes, which have shown to allow stabilization as well as targeted uptake of siRNA to cancer cells. KEYWORDS: siRNA, bioconjugate therapeutics, stabilization, specificity, therapeutic antibodies

1. INTRODUCTION 1.1. RNAi. For many diseases, the underlying causative mechanisms in protein synthesis and metabolism have been elucidated over the last decades. On the one hand, genetic defects and gene variants can contribute to the development or maintenance of a disease. On the other hand, the biochemical composition of the diseased cell usually differs from the respective normal tissue. Researchers try to use these cellular abnormalities to find a relevant target whose inhibition leads to a therapeutic success. In few instances, small molecules can fulfill these requirements, e.g., Imatinib and follow-up compounds for chronic myeloid leukemia. However, unwanted or unforeseen side effects are numerous, especially in cancer therapy. Here, also evasion strategies of tumor cell subclones often limit the efficacy of small molecules such as tyrosine kinase inhibitors. Moreover, many therapies cannot be tailored to the molecular disease factor because the identified target is judged as “undruggable” like some transcription factors and translocation products. The unavoidable switch to downstream targets often leads to less effective or less specific results. The RNA interference technology provides a new option for treatment against undruggable targets since virtually any RNA-based factor can be inhibited by small interfering RNA (siRNA). However, for systemic application the molecular structure of siRNA requires stabilization, and the specific uptake into the desired cell type should be forced by a targeting moiety. The enormous progress in the information about cellular composition, the technology of antibody design and the use of siRNA can now be combined to invent new individualized molecular treatment options. © 2017 American Chemical Society

Since its discovery by Craig C. Mello and Andrew Fire, the mechanism of RNA interference led to the development of a major tool for studying mechanics of cellular pathways by gene shutdown. Moreover, RNAi was immediately regarded as a future concept for targeted molecular therapy.1 The main advantage of the Nobel Prize awarded RNAi technology resides in the high specificity of gene silencing by complementary RNA base pairing. Furthermore, every gene can be silenced regardless of its biological function. Due to its high specificity, major knockdown of target genes is achieved with just several copies of effective siRNA molecules. Over the past decade, the rapid development of whole-genome sequencing projects and the setup of disease model databases provided the information needed to apply siRNA or cloned small-hairpin (sh) RNA molecules against disease factors. Whereas siRNA design is generally based on algorithms ensuring maximally effective knockdown efficiency,2 an algorithm predicting offtarget effects does not exist to date, which is a major challenge for the reliability of all data generated by siRNA and shRNA screens. However, achievements have been made: There is an increasing head of data showing that the use of pooled siRNAs competing for a certain gene fragment perform better with much lower tendency to cause off-target effects, a fact that was Special Issue: Bioconjugate Therapeutics Received: Revised: Accepted: Published: 1339

November 16, 2016 January 13, 2017 February 7, 2017 February 7, 2017 DOI: 10.1021/acs.molpharmaceut.6b01039 Mol. Pharmaceutics 2017, 14, 1339−1351

Review

Molecular Pharmaceutics Table 1. Phase II−III RNAi Trials disease

trial substance

RNAi target

delivery

glaucoma familial amyloidoic polyneuropathy multiple forms of cancer pancreatic cancer

QPI-1007 ALN-TTR02 FANG SiG12D LODER

CASP2 TTR furin/DC cancer vaccine KRAS G12D

topic naked siRNA IV infusion of LNP IV infusion of ex vivo electroporation surgical implantation of LODER polymer

taken up by almost all major siRNA providing companies.3 This principle was taken to the maximum with the development of enzymatically generated siRNAs (esiRNAs). For the generation of esiRNAs, computational defined stretches of genes, which exclude any repetitive or other sequences from other genes, were amplified by PCR, retranscribed to double stranded RNA, and then enzymatically digested to fragments of about 20 bp length. These pooled interfering molecules provided superior efficiency accompanied by lower off target effects, presumably caused by diminutive concentrations of the individual fragments and the tendency to compete with each other.4,5 However, although RNAi led to countless preclinical investigations and to a quicker and deeper insight into cellular regulatory mechanisms than ever before, its therapeutic application, even in early clinical trials, is still scarce and does not exceed the number of 22,6 coming in two waves: the early 2000’s and another one in 2012. Now, four approaches have entered phase III trials; see Table 1.6 1.2. siRNA Therapeutics. One of the problems that hinder siRNA as an effective therapeutic tool concerns their systemic and stable delivery, which represents a challenge especially in clinical application. Consequently, nearly half of the clinical trials rely on a topic application of naked siRNA, i.e., to the eye in age-related macular degeneration in phase II7 or glaucoma8 in phase III, inhalation in order to cure respiratory syncytial virus infection in a clinical phase IIb study.9 Despite the topical application, the use of free siRNAs still features several disadvantages: (a) hampered uptake by cells because of their strong anionic charge, (b) the short half-life of siRNA because of enzymatic attack, and (c) the chance to activate innate immune response as a result of mimicking unmethylated pathogen RNA (Figure 1A). Therefore, in the majority of the remaining trials, siRNAs were complexed by lipid nanoparticles, which provide siRNA enough stability after systemic administration by shielding them from enzymatic digestion and immune response (Figure 1B). In a slightly more elaborated concept, KRAS siRNA is complexed in a so-called Local Drug EluteR (LODER) capsule and locally implanted to a pancreatic cancer tumor mouse model. Here, siRNA is protected from degradation and is slowly released locally within the tumor for prolonged periods. This concept went into clinical trials II/III; see Table 1. However, these formulations still lack celldetermining qualities in form of targeting molecules (Figure 1B).7,10 1.3. Choice of Targeting Molecules for Target Cell Determination. The effective transport and uptake of stabilized siRNA can be achieved using selective recognition of therapeutic target cells: either ligands, which bind cellspecific surface receptors, or specifically designed antibodies raised against those receptors as well as immunopeptides. Therefore, a rapidly growing class of drugs uses targeting moieties to deliver agents to intended cells, to avoid unwanted toxicities on nonintended cells, and at the same time enrich effective agents at the site of action, the target cell;14,15 see also Figure 1C).

clinical status phase phase phase phase

II/III III III II/III

ref 8 11 12 13

1.3.1. Receptor Ligands and Peptides as Cell Determining Compounds. Ligand targeted drugs are usually of smaller molecular weight, share favorable properties in stability and half live, show good pharmacokinetics, and generally offer a lower antigenicity than antibodies.16 Therefore, ligand-targeted drugs attracted considerable attention. Several members of this class entered clinical trial phases II and III, e.g., vintafolide targeting the folate receptor17 or tTF-NGR targeting aminopeptidase N.18−21 Peptides, especially cell penetrating peptides (CPPs), were widely discussed as mediators for drug delivery.22 However, the cell-penetrating sequences still do not bear celldetermining components. At the same time, various peptides with a specific binding activity for a given cell line (celltargeting peptides, CTPs) have also been reported in the literature. Among molecules studied are the RGD peptide binding the integrins23 and the NGR peptide binding aminopeptidase N on neovasculature.18,19,24 Both consist of a sequence of only few amino acids. tTF-NGR is composed of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), which is a ligand of the surface protein aminopeptidase N (CD13). CD13 is highly overexpressed in tumor neovasculature. Binding of the NGR ligand to tumor vasculature is followed by clotting and vascular failure.18,19 Ligands carrying drugs with intracellular targets require careful considerations of drug release upon binding and internalization.25 A number of further targeting peptides were proposed for binding of tumor cells of various carcinoma or endothelium antigens with up to 12 amino acids. These targeting peptides were harnessed for siRNA transport by fusion or chemical conjugation to polycationic peptides, e.g., poly D-arginine motives 3R-12R, TAT motives, or PEN motives.26 These protein transduction domains (PTDs) exhibit high transduction ability for various cargos. Recent studies demonstrated usefulness of PTDs for gene delivery because they can form a polyplex through electrostatic interactions between positively charged PTDs and negatively charged nucleic acids. Preclinical studies focusing on cell targeting peptide-delivered siRNA therapies are currently underway27 (see also Figure 2C). 1.3.2. Antibodies and Immunoproteins as Cell Determining Compounds. The most specific biological molecule to target any protein might be an antibody. The principle of using antibodies as therapeutics was first considered by Paul Ehrlich over a century ago.28 The rapid development of the hybridoma technology in the mid 1970s enabled researchers to raise and select monoclonal antibodies (mABs) for very specific use in pathological diagnosis and as research tools.29 The antibody group is vast and yet unlimited. Antibodies have been engineered to not only find and bind the target cell molecule, but usually also to trigger a complex series of cellular mechanisms, e.g., by blocking a receptor function. This is often followed by receptor internalization. The multitude of cellular consequences ranges from receptor recycling to immune-mediated clearance or even apoptosis triggered by 1340

DOI: 10.1021/acs.molpharmaceut.6b01039 Mol. Pharmaceutics 2017, 14, 1339−1351

Review

Molecular Pharmaceutics

Figure 1. Differences in systemic administration of naked (A), stabilized (B), and (C) targeted delivery of siRNA. (A) Systemically administered naked siRNA is unprotected against enzymatic degradation, immunodetection, and renal clearance, and it is unlikely to yield therapeutic effects in target cells apart from those being directly exposed to the bloodstream, e.g., endothelium or blood cells. (B) Systemically administered siRNA complexed by carrier substances such as liposomes or nanoparticles is resistant to degradation and renal filtration and shows elongated persistence in the bloodstream and decent tissue distribution. Moreover, the coverage of the strong anionic charge enables an uptake of the siRNA-carrier conjugates to target cells, but still lacks target cell determining compounds. (C) siRNA complexed with carrier conjugates connected to targeting substances, e.g., immunoproteins or cell-specific ligands is stabilized and supports enrichment of siRNA transfer to the intended cell type, ideally excluding a vast range of other cells. 1341


Review


Figure 2. Strategies and concepts of targeted siRNA therapy. (A) Bilaminar immunoliposomes, composed of immunoprotein-decorated (green), lipid-based micelles harboring the siRNA.65−67 (B) Targeted nanoparticles consisting of transferrin-ligand (Tf) conjugated cyclodextrin-containing polycations (CDP) tailored to bind the transferrin-receptor on tumor cells and releasing the siRNA cargo.68,69 AD-PEG: adamantane (AD)-PEG conjugate. (C) Target cell-specific single-chain Fv (scFv) immunoprotein with linked light (VL) and heavy (VH) chain conjugated to a nine argininpeptide (9R) motive, which electrostatically binds the siRNA.55 (D) Genetic fusion between targeting scFv and cationic peptide protamine.70,71 (E) Target-cell receptor specific Fab fragment with linked light (VL) and heavy (VH) and parts of constant regions CL and CH chain genetically fused to protamine carries siRNA to intended cells.39 (F) Delivery conjugate composed of full IgG cell targeting antibody chemically coupled to protamine molecules binds, protects, and delivers siRNA to target cells by antibody mediated receptor internalization.37,38

antibody-dependent action or the action of conjugated effectors like toxins or radioisotopes.30−32 In therapeutic use, murine-derived mABs often raised human immune response leading to side effects and rapid clearance. Then, advanced antibody engineering led to chimeric or humanized antibodies possessing half-lives comparable to natural human IgGs.31 Although sharing disadvantages like low tissue penetration and difficulties regarding manufacturing and purification, many of these engineered antibodies entered clinical use or late clinical studies. They are falling into three major classes: (1) unmodified mABs activate immune complement mediated cytotoxicity (CMC) reactions or antibodydependent cellular cytotoxicity (ADCC), induce cell death by receptor signaling blockade, or control tumor angiogenesis. (2) Bispecific T cell engager (BiTE) and chimeric antigen receptors (CARs) link T cells to target cells.33−35 (3) Antibody−drug conjugates bind a cell-determining targeting moiety to a cytotoxic effector, i.e., a radioisotope, small molecular drug,30 or, as a novel concept, siRNAs as specific inhibitors of pathological protein synthesis36−39 (see also Figures 1C and 2F and Table 2).

Table 2. Classification of Engineered Antibodies in Clinical Usea therapeutic antibody principle unmodified mABs BiTES, bispecific T cell engangers antibody-drug conjugates

examples

ref

rituximab, cetuximab, panitumumab, bevacizumab MT110 CD3-EpCAM, blinatumomab (CD3CD19)

40−42

gemtuzumab-ozogamicin, ibritumomab-tiuxetan, bivatuzumab mertansine, inotuzumabozogamycin, ibritumomab-tiuxetan, brentuximab vedotin

45−49

35,43,44

a

Moreover, a wide range of methods were tested for siRNA stabilization and targeted delivery, as summarized in the following section.

However, in the following years it became clear that such an effective systemic administration of naked, untargeted siRNA was rather the exception than the rule. During administration process, siRNAs were exposed to enzymatic attack, innate immune response, and renal clearance. The improvement of siRNA therapeutic development went into two directions: The stabilization of siRNA by complexing agents and the integration of cell determining components into the therapeutic constructs in order to aid the effective drug conjugate directly to the intended cell type. Methods of siRNA stabilization and delivery, with or without target cell specificity, include nanoparticles and cationic liposomes52 and cationic peptides such as protamine53,54 or polyarginine.55 Naturally, the first attempts to improve on these disadvantages focused on the protection of the siRNA by incorporation

2. CELL-DETERMINING CONJUGATES FOR siRNA TRANSPORT In one of the first demonstrations of in vivo-efficiency of untargeted siRNA application, Song and colleagues used naked, i.v. administered siRNA to treat hepatitis50 (see also Figure 1A). This study based on the observation that siRNA is effectively taken up by hepatocytes, making the liver an ideal organ for siRNA based therapeutics. Also pulmonary epithelial cells and macula cells proved to be preferred target cells for naked siRNA therapy and yielded in clinical trials (Table 1).7,51 1342


Review

Molecular Pharmaceutics into liposomes56,57 (Figure 2A) or nanoparticles58−60 (Figure 2B) or by conjugation to nonpeptidic polycations such as cyclodextrin.61,62 While increasing half-live time of administered siRNA, these techniques still lack target cell determining components. Relevant methods of siRNA complexation and protection as well as examples for giving siRNA therapeutics cell determining components will be discussed in the following paragraphs. 2.1. Targeted Immunoliposomes as siRNA Carriers. Liposomes are used for transfection of mammalian cells in cell culture since the early 1980s,63,64 and lipofection still is the most applied transfection method. Hence, it is understandable that a large number of publications focused on the targeted delivery of such lipid vesicles, which in its untargeted form stabilize siRNA transport and have to be coated with a cell determining factor for targeted siRNA delivery For passing the negatively charged cytoplasmic membrane, anionic nucleic acids have been complexed by cationic substances, e.g., calcium ions in the earliest description of transfection methods, the calcium phosphate transfection.72 Consequently, for encapsulation in liposomes, cationic lipids were utilized. They consist of lipids with positively charged headgroup (Figure 2A) and can combine on their own or in conjunction with neutral lipids to form small unilamellar vesicles. Nucleic acids can interact spontaneously with these vesicles and form lipid−DNA complexes from the positively charged group on the cationic lipid with the negatively charged phosphate groups on the DNA. Transfection by liposomeentrapped nucleic acids, or lipofection, is an effective gene delivery method for a variety of cell types. The resulting lipoplexes, however, are causing considerable cellular toxicity and therefore are a constant subject of optimization in applicability. A few well-characterized cationic lipids used to form lipoplexes are 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), [N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium methyl sulfate] (DOTAP), 3β[N(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DCChol), and dioctadecylamidoglycylspermine (DOGS).73 Dioleoylphosphatidylethanolamine (DOPE) is a neutral helper lipid often used in conjunction with cationic lipids because of its membrane-destabilizing effects at low pH, which facilitates endosomal escape.74 The combination of cationic liposomes with polyethylene glycol (PEG) enhances blood circulation time by restriction of uptake in the reticuloendothelial system, a decreased degradation by metabolic enzymes and a reduction of immunogenicity.75 The study of Zhang et al. in 200465 was among the first to show integration of antibodies as cell determining compound into PEGylated liposomes to treat a xenografted brain cancer model in Scid mice. Here, two independent monoclonal antibodies against the transferrin receptor of the mouse endothelium mTfR and the insulin receptor HIR were used (a) to break the blood−brain barrier (BBB) by transcytosis of the cancer vascular endothelium and (b) internalize into the xenografted U87 human gliomas by insulin-receptor mediated action of the HIR mAB. The RNAi effector was not a siRNA, but a relatively large shRNA plasmid containing RNAi sequences targeting the EGF receptor, which was incorporated into the double-targeted immunoliposome. The knock-down of EGFR in mouse brain sections was shown in immunofluorescence as well as a reduction in vascular density, and the authors assessed a dramatic increase in survival time in mice with xenografted glioma cells treated with the targeted RNAi

conjugate versus saline, which is a stunning result for this advanced bifunctional targeting strategy. However, this technique so far was never used in any clinical study. PEGylated immunoliposomes were also applied to treat an experimental murine melanoma-lung metastasis model in mice.67 Here, human single chain Fv fragments (scFv) as cell determining compound were identified to detect and internalize into murine and human cells, presumably by receptor internalization. These scFv conjugated to self-assembling formulations of DOTAP and cholesterol loaded with siRNAs complexed by protamine. The method consisted of chemical cross-linking of the cysteine-bearing scFv with the Mal-PEGDSP, a phospholipid PEG conjugate, which integrated to the liposomes by hydrophobic interaction.76 In the conceptual study, siRNA versus c-myc and MDM2 was used to treat mice bearing melanoma-induced metastasis systemically. The RNAi effect resulted in repression of c-myc expression as well as the negative regulator of the p53 tumor suppressor MDM2. Metastasis of luciferase-tagged melanoma cells was inhibited by about 70%. Furthermore, the authors tried to interfere with MAPK signaling by systemic delivery or miRNA-34a.77 Decoration of siRNA-stabilizing liposomes with immune fragments for specific cell targeting was also done with antiHER2-specific Fab fragments coupled to Mal-GEG-DSP by thiol-activation of the Fab.78 Here, the authors paid much attention to control the size of liposomes. A series of extruding treatments was performed with decreasing pore sizes of filters to form defined unilamellar vesicles from multilamellar structures. Then, uncomplexed siRNA was loaded to the liposomes by a lyophylization and rehydration procedure. In a follow up-publication, the same group optimized the physicochemical properties of the above concept to deliver Cy5-tagged, protamine-complexed siRNA to MDA-MB 231 breast cancer xenografted tumors in mice. The targeted versus nontargeted delivery triggered by an anti-EGFR Fab fragment resulted in a remarkable enrichment of cy5-siRNA in the EGFR-expressing tumor versus organ background.79 Together with further modifications, the same setup was applied for EGFR expressing hepatocarcinoma cells80 and xenografts.66 Immunoliposomes were not only used to treat cancer cells but also infectious diseases: PEGylated DC-Chol-Dope decorated with an H5N1 avian influenza virus hemagglutinin specific single chain Fv antibody fragment was used to treat H5N1-infected MDCK cells, and antiviral siRNAs specifically reduced virus titers.81 2.2. Targeted Nanoparticles as siRNA Carriers. Nanoparticles (NPs) led to significant interest over the past decade, namely, in cancer therapy. Here, they can overcome limitations of conventional chemotherapy. Because of their conceptual heterogeneity, NPs can be formed in a range of sizes (1−1000 nm) and be produced using materials including polymers (for instance, biodegradable polymeric nanoparticles or dendrimers), lipids (e.g., solid−lipid nanoparticles, liposomes, see section 2.1), and others (Figure 2B). In 2005, a concept using cyclodextrin-containing polycations (CDP) was introduced, where a mechanism of self-assembling with siRNA to stabilize was shown to form colloidal particles of about 50 nm in diameter, and their terminal imidazole groups act as proton sponges to assist in the intracellular trafficking and release of the nucleic acid.69 Here, CDP protects the siRNA from degradation (Figure 2B). The targeting moiety used here was transferrin to target the transferrin receptor, which was highly expressed on Ewing sarcoma cell lines in this 1343


Review


Figure 3. Methods of conjugation between cell determining immunoproteins and siRNA complexing agents. (A) Chemical conjugation of cell determining IgG molecule with siRNA complexing cationic peptide protamine by means of bispecific linker sulfo-SMCC. Amino functions in protamine were conjugated to activated sulfo SMCC and then to cysteines of the immunoglobulin backbone.38 (B) Biotin−avidin conjugation of immunglobulin and protamine. First protamine is biotinylated and then coupled to monovalent IgG activated with avidin.53 (C) Disulfide bond formation by oxidative coupling of arginine peptide-SH as siRNA carrier and scFv-SH as cell determining agent.55 (D) Use of biotinylated siRNA coupled to streptavidin-activated mAb.91 (E) Direct covalent conjugation of siRNA and carrier by means of advanced chemical conjugation methods.92 1344


Review


mild reduction materials can be used to reduce interchain disulfide bonds or additional cysteine residues can be inserted in the antibody molecule by means of genetic engineering (Figure 3A). However, the cysteine redox state and cysteine availability of a choice of different targeting antibodies can vary significantly and can even vary between individual preparations. Hence, one has to control the prevalence of free conjugation cysteine residues or expose them by controlled reduction without destroying antibody targeting efficiency.88,90 In recombinant therapeutic IgG1 antibody stored in specific stabilizing solutions such as commercially cetuximab, we discovered approximately 0.5−2 reduced cysteines per mol of antibody judged by Ellmans test. This ratio can be greatly enhanced by controlled partial reduction with Tris(2carboxyethyl)-phosphin (TCEP). Intensive reduction with TCEP will induce formation of monovalent mABs or even HC and LC fragments.89 However, mouse monoclonal antibodies can offer much more reduced cysteines for advanced chemical manipulations,90 even in steady state. The above cited literature proves that protamine fusion proteins are a powerful tool for cell specific siRNA targeting; however, the coupling of protamine peptides to the targeting moiety is limited by the genetic fusion only allowing N- or Cterminal fusion and hence usually only one molecule of protamine per targeting moiety, thereby limiting the siRNA coupling ratio. Hauser and colleagues53 followed another concept: They connected protamine not by genetic fusion, but cleaved a targeting IgG by partial reduction of disulfide bridges and chemically introduced an avidin molecule of the available cysteine sulfhydryl function (Figure 3B). Protamine, in turn, was marked with biotin, enabling a stable connection targeting moiety−S-avidin−biotin−protamine. With this conceptual conjugate, murine podoytes were targeted by application of nephrin and TRPC6 siRNA. The conjugates reduced the glomerulus-located proteins in vivo. Although the exact composition of the targeting conjugate has not been characterized and elucidated here, the combined chemical coupling strategy discloses the capabilities and the potential of chemical conjugation. Our own contributions to the design of an effective siRNA carrier antibody complex (Figure 3A) were inspired by the conjugation concept first published by Kumar et al.,55 exhibiting a strategy of expressed protein ligation (see also Figure 2C). They expressed a CD7 specific scFv antibody with a C-terminal cysteine and coupled this targeting moiety by auto-oxidative disulfide formation to a cysteine-containing nona-D-arginine synthetic peptide as a siRNA carrier (Figure 3C). The resulting conjugate was tested for proper internalization with the CD7 receptor. Loaded with antiviral siRNA, it suppressed viral replication in NOD-scid mice reconstituted with HIV-infected human lymphocytes. However, the introduction of terminal cysteines resembles numerous technical disadvantages in protein expression, e.g., precipitation in bacterial expression and unwanted dimerization in mammalian cell culture expression. Furthermore, the expressed ligation of reduced cysteines in trans between antibody and peptide carrier is difficult to control and was not practicable in our hands. Consequently, we used bispecific chemical linkers such as sulfo-SMCC to activate protamine N-terminally and conjugate it to available cysteine residues in the cetuximab antibody backbone38 (Figures 2F and 3A). The consecutive antibody− protamine conjugate displays a robust molecular weight shift suggesting conjugation of mAB to more than two molecules of

case. Application of these compounds coupled to EWS-FLI1 central oncogene-specific siRNA achieved transient tumor growth reduction in xenografted NOD-Scid mice. The same CDP delivery concept was applied to solid tumors in 2010,68 to nonhuman primates,82 and resulted in a first-in-man study with nanoparticles CALAA-01.83 Although the full potential of CALAA-01 was not evaluated in this phase Ia/Ib clinical study, CALAA-01 was well tolerated during the initial dose escalation, but the substance showed enhanced renal clearance. 2.3. Harnessing Protamine for Targeted Delivery of siRNAs. Many attempts have been made to couple a targeting moiety for specific cell determination such as an antibody or antibody fragment to a siRNA carrier substance for its stabilization such as protamine by means of genetic fusion. This seems a favorable method because of the clear control over the immunoprotein−protamine conjugate ratio; however, it limits the ratio to 1:1 and so determines the siRNA carrier activity. Protamine is a cationic, nucleic acid-binding peptide transporting a complete set of genomic DNA compressed in the sperm-head. Since it is able to complex nucleic acids and to facilitate the transition of nucleic acids across the cytoplasmic membrane, this attracted numerous researchers to study application in transfection, targeted delivery, and gene therapy.54,76,84−86 It was tested as a nucleic acid delivery vehicle and connected to various cell determining targeting moieties. In 2005 Song et al.39 presented a genetic fusion protein connecting a Fab fragment (F105) against a human immunodeficiency virus HIV gp 160 envelope protein and a shortened protamine peptide (Figure 2E). The fusion protein complexed siRNAs targeting HIV gag protein, and the conjugate was able to target hard-to-transfect HIV infected T cells and HIV envelope transfected melanoma cells. The siRNA−F105 carrier conjugate inhibited HIV replication in infected T cells. To verify the targeting principle, the same strategy was applied to target ErbB2 by Erb2 single chain antibody fused to protamine. This antibody conjugate was coupled to Ku 70 siRNA87 and reduced Ku70 expression only in ErbB2 expressing cells, but not in cells without ErbB2 expression. This clearly indicates a receptor-dependent mechanism of siRNA targeting. In another publication, the genetic single chain-Fv-protamine fusion principle was also adopted for targeting lymphocytes by lymphocyte function associated antigen (LFA-1) directed scFv-protamine coupled to siRNA also silencing Ku70 in xenografted K562 cells.71 Moreover, the authors demonstrated Ku70 silencing in mixed populations of LFA-1 expressing and nonexpressing cells selectively effective in the cells possessing the LFA-1. This indicates a mechanism of siRNA delivery dependent on the cell surface antigen expression. 2.4. Conjugation Methods, Linkers, and Examples. Naturally, in a bioconjugate, the constraints regarding targeting moiety and payload binding module are very strict. So the optimization steps on pharmacotypic properties of a given conjugate often are achieved by subtle modifications of the linking unit. Most important requirements are that the linker should never sterically interfere with target molecule binding or payload binding or release. Therefore, the design of the conjugation method may hugely affect drug performance. In general, the process of chemical modification of proteins is an important concept to probe natural systems and create novel therapeutic proteins.88 In the antibody−drug conjugate field, conjugation processes often rely on cysteine modification methods.89 In an antibody, 1345


Review


Figure 4. Vesicular trafficking and endosomal escape of receptor-targeted drug conjugates. The binding of an immunoconjugate to the desired receptor results in internalization by endocytosis. The compartment is constricted in early endosomes, which are converted to late endosomes by further acidification. Certain vesicles are recycled to the membrane and depending on the mechanism of action, part of the late endosomes are ruptured and the content containing the siRNAis liberated to the cytoplasm. Fluorescence micrograph show RD rhabdomysarcoma cells expressing EGFR target with Alexa488-tagged siRNA by anti-EGFR (Cetuximab)−protamine carrier system (Bäumer et al., manuscript in preparation).

protamine.37 The resulting conjugate binds multiple molecules of siRNA per molecule of carrier and transports them to the intended target cells in vivo. Chemical conjugation of protamine did not interfere with target−receptor interaction of the targeting antibody Cetuximab, the conjugate internalized to cells, and siRNA was detectable in intracellular vesicular structures. Especially the possibility to increase the number of protamines per mol of targeting antibody makes this strategy intriguing and furthermore allows for composition as a building-block concept, where the directing antibody can simply be exchanged by another that is more suitable to interfere with certain receptors or cell surface molecules on the target cells. First data for this modular use of the concept have been published just recently.37 Completely different strategies to couple siRNA to an antibody are conjugations based on the streptavidin/biotin couple. Xia and coauthors used biotinylated siRNA and bound them to monoclonal antibody to insulin receptor (HIRMaB) genetically fused to streptavidin (SA) and transfected cells in vitro with this substance conjugate91 (Figure 3D). However, the close-to-covalent binding affinity between antibody−SA and biotin−siRNA makes a liberation of both in endosomal vesicles difficult. Other research groups went even further and covalently bound chemically activated siRNAs to cysteines on an anti-CD71 Fab fragment (Figure 3E). Although the process of liberation of siRNA is not clear here either, the authors stated robust silencing effect in target muscle tissues lasting for up to one month.92 This may result from the fact that the overall molecular weight of the antibody−siRNA couple is relatively low compared with other types of conjugation methods, nanoparticles, and liposomes. The probability of large complex formation without cationic electrostatic binding moieties like protamine, PEI, or cyclodextrin is low, and it may favor endosomal release, although the cargo to carrier ratio is definitely limited to 1:1 here.

3. INTRACELLULAR TRAFFICKING PATHWAY: ENDOSOMAL ESCAPE Despite the improvements in targeted delivery concepts, the intracellular fate of delivered agents remains a central issue, especially if the delivered cargo is sensitive to enzymatic destruction in vesicular pathways like siRNA molecules. Here, the endocytotic pathway is referred to be the major uptake mechanism for biological agents such as receptor-directed antibodies, other proteins or peptides, as well as DNA and siRNA.74,85 The process of endocytosis entails an inward budding of the cytoplasmic membrane, and while entrapped in early endosomes, the included cargo (e.g., internalized receptors activated by ligand binding; see Figure 4) are sorted for recycling or lysosomal digest. The latter late endosomal vesicles are acidified (pH 5−6) by membrane-bound protonpump ATPases. These late endosomes are relocated to the lysosomes, which are further acidified (pH 4−5) and contain various nucleases and proteases that degrade nucleic acids and proteins. Consequently, the evasion of lysosomal digest depicts a limiting step in achieving an effective biological therapy. The process of evasion is the so-called endosomal escape pathway, which can be triggered or enhanced by basically two events, the pH-buffering proton sponge effect and fusions with the endosomal membrane.74 The so-called proton sponge effect occurs when materials of a high buffering capacity are entrapped in an endosome. The massive influx of H+ protons and the resulting coinflux of water causes an endosomal swelling and rupture of the endosomal membrane. Proteins with high content of tertiary amines are an example, namely, histidine rich molecules and various cationic polymers such as poly(ethylenimine) (PEI)93 or poly(amidoamine) (PAA.).94,95 Also, buffering components such as chloroquine and methylamine do the same effect.96 Viral proteins often show peptide domains that have functions, which enhance endosomal escape by fusion with the endosomal membrane. Examples were influenza virus hemagglutinin N-terminal peptide that under low pH 1346


Review


regarding optimization of endosomal release of drug conjugate and cargo. Last, there are the direct covalently linked carrier-tosiRNA conjugates, which display the smallest sized carrier molecules. Although this class naturally is the most stable and least prone to disintegration in the bloodstream and tissue, this fact might be counterproductive because it hinders liberation of RNAi molecules form the carrier in the target cell. In general, a superior serum half-life time combined with a good biodistribution pattern is highly desirable in all kinds of targeted drug conjugates, and that goes not only for leaky tumor vasculature. As an example, high molecular weight full IgGs show better serum half life time but lower tissue penetration than smaller immunoconstructs, an inverse relation that can be transferred to more complex composed targeted nanoparticles and liposomes.108 More work has to be done to reveal the fine balancing checks of stability versus availability in targeted drug delivery, especially the delivery of siRNAs.

conditions forms secondary structure conferring fusion with the endosomal membrane, which then leads to early endosomal escape. Also, deduced peptides from this region, the KALA sequence97−99 and the GALA sequence,100,101 are amphiphilic peptides and induce endosomal rupture by pH-induced conformational change enabling a fusion with the endosomal membrane. A second peptide derived from influenza virus, the peptide diINF7, enhanced endosomal escape combined with lipid carriers for siRNA,102 protein,103 and gene delivery.104 In addition, a number of arginine-rich peptides are known as strong enhancers of cellular transfection with siRNAs, DNA, or proteins by promoting binding of proteoglycans on the cell surface and destabilization of endosomal membranes by fusion. Although being one of the best studied in delivery, the endosomal escape route of the protamine peptide is not clear. Protamine condenses DNA in the sperm and consists of nearly 70% arginine. Protamine has been widely used in delivery of siRNAs and DNA.37,39,53,54 Proteolytic digested peptides from protamine-sulfate (LMW-protamine, LMWP) possess a defined VSRRRRRRGGRRRR sequence and shows cell penetrating activities (CPP), lower immunogenicity, and enhanced gene delivery activity.54,86 The high arginine content may contribute to endosomal rupture by proton sponge effect. However, the endosomal escape of protamine-related peptides can be enhanced by fusion to various viral peptides and proteins.

5. CONCLUSIONS With a number of clinical trials in late-stage phase, the field of RNAi therapy progressed well over the past decade. Therefore,

4. COMPARING CONCEPTS OF ANTIBODY-MEDIATED siRNA DELIVERY Generally, the strategies of cell-specific RNAi can be divided in four different concepts: (A) Cationic lipid composite vesicles modified by PEGylation and decorated with targeting proteins such as immunoproteins or ligands, loaded with siRNA or shRNA. (B) Nanoparticles composed of substances like cyclodextrin-containing polycations (CDP), also secondary modified, loaded with RNAi molecule and decorated with targeting moieties as above. (C) Genetic or chemical fusions between dedicated cationic siRNA carrier peptides like protamine and guiding proteins, usually antibodies or deduced fragments, which can be conjugated with siRNA. (D) Direct covalent or interaction linkage between siRNA and targeting protein. All of them have their advantages and disadvantages: high molecular weight immunoliposomes show superior siRNA protection in vivo and show long persistence in the bloodstream, but tissue penetration is clearly an issue with such large constructs. Furthermore, their production is laborious, complex, and prone to difficulties although being one of the best studied to date. Targeted nanoparticles somewhat share the same pros and cons, but seem less complex in production. siRNA-carrier-guide conjugates attracted considerable attraction and are less complex in construction and production. Because of their limited molecular weight, they may show benefits in tissue penetration while avoiding renal and immunogenic clearance. In this subgroup, the protamineconjugated carriers attracted most attention, divided in genetically fused but also chemically conjugated molecules. Protamine has a long-standing medical use as a heparin antidote105 and insulin retarding material84 and is approved by the FDA for these uses. Furthermore, its known ability to complex nucleic acids triggered very early discussions about its use in human gene therapy.106,107 Considering all these experiences over a long time, protamine might be a good candidate for the formulation of an effective siRNA delivery− drug conjugate, but maybe more attention has to be paid

Figure 5. Adjusting dual specificities in targeted siRNA therapeutic scenario. A highly exclusive siRNA target, e.g., an oncogenic fusion gene only present in a population of tumor-initiating cells can be treated with a siRNA guided by a fairly nonspecific targeting moiety only ensuring a mild enrichment of siRNA in the tumor cell (left-hand side). However, the delivery of a much less cell-determining siRNA such as a housekeeping-gene-silencing siRNA requires a very high degree of specificity regarding the exclusive delivery (right-hand side). Anyway, cell-targeted siRNA therapies should always provide a dual specificity ensuring a high degree of enrichment in the desired cell type and the most cell-determining interference with gene expression that is possible.

approval by the FDA will be likely for a member of these calls of drugs within the next years. This breakthrough then will also help to pave the way for other RNAi drugs. Very likely, the definition of new and effective RNAi target genes will improve over the next years, and effort has to be spent in limiting offtarget effects. For the most important improvement necessary in the development of delivery strategies, we will need robust and cell-specific carrier systems of limited molecular weight to ensure good cycling times and tissue penetration in order to reach less accessible areas. The highest degree of therapeutic stringency will be reached if a cellular-exclusive and diseaseinitiating gene is silenced specifically in those cells by a strict choice of delivery component, limiting RNAi effect to only these cells. The lower the RNAi selectivity is, regarding the biodistribution of the target gene expression, the higher the delivery specificity must be and vice versa (Figure 5). This is important to exclude unwanted side effects, e.g., by silencing 1347


Review


(9) DeVincenzo, J.; Lambkin-Williams, R.; Wilkinson, T.; Cehelsky, J.; Nochur, S.; Walsh, E.; Meyers, R.; Gollob, J.; Vaishnaw, A. A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 8800−8805. (10) Tabernero, J.; Shapiro, G. I.; LoRusso, P. M.; Cervantes, A.; Schwartz, G. K.; Weiss, G. J.; Paz-Ares, L.; Cho, D. C.; Infante, J. R.; Alsina, M.; Gounder, M. M.; Falzone, R.; Harrop, J.; White, A. C.; Toudjarska, I.; Bumcrot, D.; Meyers, R. E.; Hinkle, G.; Svrzikapa, N.; Hutabarat, R. M.; Clausen, V. A.; Cehelsky, J.; Nochur, S. V.; GambaVitalo, C.; Vaishnaw, A. K.; Sah, D. W.; Gollob, J. A.; Burris, H. A., 3rd First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discovery 2013, 3, 406−417. (11) Coelho, T.; Adams, D.; Silva, A.; Lozeron, P.; Hawkins, P. N.; Mant, T.; Perez, J.; Chiesa, J.; Warrington, S.; Tranter, E.; Munisamy, M.; Falzone, R.; Harrop, J.; Cehelsky, J.; Bettencourt, B. R.; Geissler, M.; Butler, J. S.; Sehgal, A.; Meyers, R. E.; Chen, Q.; Borland, T.; Hutabarat, R. M.; Clausen, V. A.; Alvarez, R.; Fitzgerald, K.; GambaVitalo, C.; Nochur, S. V.; Vaishnaw, A. K.; Sah, D. W.; Gollob, J. A.; Suhr, O. B. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 2013, 369, 819−829. (12) Nemunaitis, J.; Barve, M.; Orr, D.; Kuhn, J.; Magee, M.; Lamont, J.; Bedell, C.; Wallraven, G.; Pappen, B. O.; Roth, A.; Horvath, S.; Nemunaitis, D.; Kumar, P.; Maples, P. B.; Senzer, N. Summary of bi-shRNA/GM-CSF augmented autologous tumor cell immunotherapy (FANG) in advanced cancer of the liver. Oncology 2014, 87, 21−29. (13) Zorde Khvalevsky, E.; Gabai, R.; Rachmut, I. H.; Horwitz, E.; Brunschwig, Z.; Orbach, A.; Shemi, A.; Golan, T.; Domb, A. J.; Yavin, E.; Giladi, H.; Rivkin, L.; Simerzin, A.; Eliakim, R.; Khalaileh, A.; Hubert, A.; Lahav, M.; Kopelman, Y.; Goldin, E.; Dancour, A.; Hants, Y.; Arbel-Alon, S.; Abramovitch, R.; Shemi, A.; Galun, E. Mutant KRAS is a druggable target for pancreatic cancer. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 20723−20728. (14) Bartlett, D. W.; Su, H.; Hildebrandt, I. J.; Weber, W. A.; Davis, M. E. Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 15549−15554. (15) Yu, B.; Zhao, X.; Lee, L. J.; Lee, R. J. Targeted delivery systems for oligonucleotide therapeutics. AAPS J. 2009, 11, 195−203. (16) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents. Nat. Rev. Drug Discovery 2015, 14, 203−219. (17) Vergote, I.; Leamon, C. P. Vintafolide: a novel targeted therapy for the treatment of folate receptor expressing tumors. Ther. Adv. Med. Oncol. 2015, 7, 206−218. (18) Stucke-Ring, J.; Ronnacker, J.; Brand, C.; Holtke, C.; Schliemann, C.; Kessler, T.; Schmidt, L. H.; Harrach, S.; Mantke, V.; Hintelmann, H.; Hartmann, W.; Wardelmann, E.; Lenz, G.; Wunsch, B.; Muller-Tidow, C.; Mesters, R. M.; Schwoppe, C.; Berdel, W. E. Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction. Oncotarget 2016, DOI: 10.18632/oncotarget.12559. (19) Brand, C.; Schliemann, C.; Ring, J.; Kessler, T.; Baumer, S.; Angenendt, L.; Mantke, V.; Ross, R.; Hintelmann, H.; Spieker, T.; Wardelmann, E.; Mesters, R. M.; Berdel, W. E.; Schwoppe, C. NG2 proteoglycan as a pericyte target for anticancer therapy by tumor vessel infarction with retargeted tissue factor. Oncotarget 2016, 7, 6774− 6789. (20) Bieker, R.; Kessler, T.; Schwoppe, C.; Padro, T.; Persigehl, T.; Bremer, C.; Dreischaluck, J.; Kolkmeyer, A.; Heindel, W.; Mesters, R. M.; Berdel, W. E. Infarction of tumor vessels by NGR-peptide-directed targeting of tissue factor: experimental results and first-in-man experience. Blood 2009, 113, 5019−5027. (21) Dreischaluck, J.; Schwoppe, C.; Spieker, T.; Kessler, T.; Tiemann, K.; Liersch, R.; Schliemann, C.; Kreuter, M.; Kolkmeyer, A.; Hintelmann, H.; Mesters, R. M.; Berdel, W. E. Vascular infarction

common survival factors in different cells with possible fatal consequences. Cellular specific delivery systems will enable us to interfere with central pathway target genes without doing harm to nonintended cells. Under some preconditions, it also may be necessary to interfere with expression of more than one gene in the target cells. Delivery systems of the future should take this into account and provide effective transport of many molecules of RNAi per molecule of carrier. In combination, this will be a great opportunity to reach hard-to-target diseases.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sebastian Bäumer: 0000-0002-2790-1410 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Wilhelm Sander Stiftung grant nos. 2009.041.2 (to S.B.), 2014.054.1 (to W.E.B. and S.B.), and 2010.015.1 (to W.E.B.); by Deutsche Forschungsgemeinschaft, DFG EXC 1003 Cells in Motion, Cluster of Excellence (to W.E.B.); by Deutsche Krebshilfe 70112282 to S.B. and N.B. This work was also supported by the funds “Innovative Medical Research“ of the University of Münster Medical School (IMF) grant nos. 111418 and 211502 (to S.B.), 121314 (to N.B.), and 111501 (to N.B. and S.B.) by the Medical Faculty, University of Münster. The authors like to thank Neele Appel and Lisa Terheyden for contributing data to Figure 4.

■

REFERENCES

(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 1998, 391, 806−811. (2) Reynolds, A.; Leake, D.; Boese, Q.; Scaringe, S.; Marshall, W. S.; Khvorova, A. Rational siRNA design for RNA interference. Nat. Biotechnol. 2004, 22, 326−330. (3) Parsons, B. D.; Schindler, A.; Evans, D. H.; Foley, E. A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One 2009, 4, e8471. (4) Surendranath, V.; Theis, M.; Habermann, B. H.; Buchholz, F. Designing efficient and specific endoribonuclease-prepared siRNAs. Methods Mol. Biol. 2013, 942, 193−204. (5) Kittler, R.; Surendranath, V.; Heninger, A. K.; Slabicki, M.; Theis, M.; Putz, G.; Franke, K.; Caldarelli, A.; Grabner, H.; Kozak, K.; Wagner, J.; Rees, E.; Korn, B.; Frenzel, C.; Sachse, C.; Sonnichsen, B.; Guo, J.; Schelter, J.; Burchard, J.; Linsley, P. S.; Jackson, A. L.; Habermann, B.; Buchholz, F. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat. Methods 2007, 4, 337−344. (6) Bobbin, M. L.; Rossi, J. J. RNA Interference (RNAi)-Based Therapeutics: Delivering on the Promise? Annu. Rev. Pharmacol. Toxicol. 2016, 56, 103−122. (7) Kaiser, P. K.; Symons, R. C.; Shah, S. M.; Quinlan, E. J.; Tabandeh, H.; Do, D. V.; Reisen, G.; Lockridge, J. A.; Short, B.; Guerciolini, R.; Nguyen, Q. D. Sirna-027 Study Investigators RNAibased treatment for neovascular age-related macular degeneration by Sirna-027. Am. J. Ophthalmol. 2010, 150, 33−39.e2. (8) Solano, E. C.; Kornbrust, D. J.; Beaudry, A.; Foy, J. W.; Schneider, D. J.; Thompson, J. D. Toxicological and pharmacokinetic properties of QPI-1007, a chemically modified synthetic siRNA targeting caspase 2 mRNA, following intravitreal injection. Nucleic Acid Ther. 2014, 24, 258−266. 1348


Review

Molecular Pharmaceutics by subcutaneous application of tissue factor targeted to tumor vessels with NGR-peptides: activity and toxicity profile. Int. J. Oncol. 2010, 37, 1389−1397. (22) Bolhassani, A. Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochim. Biophys. Acta, Rev. Cancer 2011, 1816, 232−246. (23) Tian, H.; Lin, L.; Chen, J.; Chen, X.; Park, T. G.; Maruyama, A. RGD targeting hyaluronic acid coating system for PEI-PBLG polycation gene carriers. J. Controlled Release 2011, 155, 47−53. (24) Corti, A.; Curnis, F.; Rossoni, G.; Marcucci, F.; Gregorc, V. Peptide-mediated targeting of cytokines to tumor vasculature: the NGR-hTNF example. BioDrugs 2013, 27, 591−603. (25) Allen, T. M. Ligand-targeted therapeutics in anticancer therapy. Nat. Rev. Cancer 2002, 2, 750−63. (26) Ren, Y.; Hauert, S.; Lo, J. H.; Bhatia, S. N. Identification and characterization of receptor-specific peptides for siRNA delivery. ACS Nano 2012, 6, 8620−8631. (27) Jarver, P.; Coursindel, T.; Andaloussi, S. E.; Godfrey, C.; Wood, M. J.; Gait, M. J. Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA. Mol. Ther.–Nucleic Acids 2012, 1, e27. (28) Ehrlich, P. Collected Studies on Immunity; John Wiley & Sons: New York, 1906. (29) Weiner, L. M.; Surana, R.; Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 2010, 10, 317−327. (30) Casi, G.; Neri, D. Antibody-drug conjugates: basic concepts, examples and future perspectives. J. Controlled Release 2012, 161, 422− 428. (31) Weiner, G. J. Building better monoclonal antibody-based therapeutics. Nat. Rev. Cancer 2015, 15, 361−370. (32) Lambert, J. M. Drug-conjugated antibodies for the treatment of cancer. Br. J. Clin. Pharmacol. 2012, 76, 248. (33) Singer, H.; Kellner, C.; Lanig, H.; Aigner, M.; Stockmeyer, B.; Oduncu, F.; Schwemmlein, M.; Stein, C.; Mentz, K.; Mackensen, A.; Fey, G. H. Effective elimination of acute myeloid leukemic cells by recombinant bispecific antibody derivatives directed against CD33 and CD16. J. Immunother. 2010, 33, 599−608. (34) Pule, M. A.; Savoldo, B.; Myers, G. D.; Rossig, C.; Russell, H. V.; Dotti, G.; Huls, M. H.; Liu, E.; Gee, A. P.; Mei, Z.; Yvon, E.; Weiss, H. L.; Liu, H.; Rooney, C. M.; Heslop, H. E.; Brenner, M. K. Virusspecific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat. Med. 2008, 14, 1264−1270. (35) Bargou, R.; Leo, E.; Zugmaier, G.; Klinger, M.; Goebeler, M.; Knop, S.; Noppeney, R.; Viardot, A.; Hess, G.; Schuler, M.; Einsele, H.; Brandl, C.; Wolf, A.; Kirchinger, P.; Klappers, P.; Schmidt, M.; Riethmuller, G.; Reinhardt, C.; Baeuerle, P. A.; Kufer, P. Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 2008, 321, 974−977. (36) Palanca-Wessels, M. C.; Booth, G. C.; Convertine, A. J.; Lundy, B. B.; Berguig, G. Y.; Press, M. F.; Stayton, P. S.; Press, O. W. Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells. Oncotarget 2016, 7, 9561−9575. (37) Bäumer, N.; Appel, N.; Terheyden, L.; Buchholz, F.; Rossig, C.; Muller-Tidow, C.; Berdel, W. E.; Baumer, S. Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown. Nat. Protoc. 2016, 11, 22−36. (38) Bäumer, S.; Bäumer, N.; Appel, N.; Terheyden, L.; Fremerey, J.; Schelhaas, S.; Wardelmann, E.; Buchholz, F.; Berdel, W. E.; MullerTidow, C. Antibody-Mediated Delivery of Anti-KRAS-siRNA In Vivo Overcomes Therapy Resistance in Colon Cancer. Clin. Cancer Res. 2015, 21 (6), 1383−94. (39) Song, E.; Zhu, P.; Lee, S. K.; Chowdhury, D.; Kussman, S.; Dykxhoorn, D. M.; Feng, Y.; Palliser, D.; Weiner, D. B.; Shankar, P.; Marasco, W. A.; Lieberman, J. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 2005, 23, 709−17.

(40) Feuring-Buske, M.; Buske, C.; Unterhalt, M.; Hiddemann, W. Recent advances in antigen-targeted therapy in non-Hodgkin’s lymphoma. Ann. Hematol. 2000, 79, 167−174. (41) Kies, M. S.; Harari, P. M. Cetuximab (Imclone/Merck/BristolMyers Squibb). Curr. Opin. Investig Drugs 2002, 3, 1092−1100. (42) Langerak, A.; River, G.; Mitchell, E.; Cheema, P.; Shing, M. Panitumumab monotherapy in patients with metastatic colorectal cancer and cetuximab infusion reactions: a series of four case reports. Clin. Colorectal Cancer 2009, 8, 49−54. (43) Cioffi, M.; Dorado, J.; Baeuerle, P. A.; Heeschen, C. EpCAM/ CD3-Bispecific T-cell engaging antibody MT110 eliminates primary human pancreatic cancer stem cells. Clin. Cancer Res. 2012, 18, 465− 474. (44) Feucht, J.; Kayser, S.; Gorodezki, D.; Hamieh, M.; Doring, M.; Blaeschke, F.; Schlegel, P.; Bosmuller, H.; Quintanilla-Fend, L.; Ebinger, M.; Lang, P.; Handgretinger, R.; Feuchtinger, T. T-cell responses against CD19+ pediatric acute lymphoblastic leukemia mediated by bispecific T-cell engager (BiTE) are regulated contrarily by PD-L1 and CD80/CD86 on leukemic blasts. Oncotarget 2016, DOI: 10.18632/oncotarget.12357. (45) Castaigne, S.; Pautas, C.; Terre, C.; Raffoux, E.; Bordessoule, D.; Bastie, J. N.; Legrand, O.; Thomas, X.; Turlure, P.; Reman, O.; de Revel, T.; Gastaud, L.; de Gunzburg, N.; Contentin, N.; Henry, E.; Marolleau, J. P.; Aljijakli, A.; Rousselot, P.; Fenaux, P.; Preudhomme, C.; Chevret, S.; Dombret, H. Acute Leukemia French Association Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): a randomised, openlabel, phase 3 study. Lancet 2012, 379, 1508−1516. (46) Gleissner, B.; Schlenk, R.; Bornhauser, M.; Berdel, W. E. Gemtuzumab ozogamicin (mylotarg) for the treatment of acute myeloid leukemia–ongoing trials. Oncol. Res. Treat. 2007, 30, 657− 662. (47) Hoelzer, D. Targeted therapy with monoclonal antibodies in acute lymphoblastic leukemia. Curr. Opin. Oncol. 2013, 25, 701−706. (48) Rizzieri, D. Zevalin((R)) (ibritumomab tiuxetan): After more than a decade of treatment experience, what have we learned? Crit. Rev. Oncol. Hematol. 2016, 105, 5−17. (49) Ramsey, S. D.; Nademanee, A.; Masszi, T.; Holowiecki, J.; Abidi, M.; Chen, A.; Stiff, P.; Viviani, S.; Sweetenham, J. W.; Radford, J.; Zhu, Y.; Bonthapally, V.; Thomas, E.; Richhariya, A.; Hunder, N. N.; Walewski, J.; Moskowitz, C. H. Quality of life results from a phase 3 study of brentuximab vedotin consolidation following autologous haematopoietic stem cell transplant for persons with Hodgkin lymphoma. Br. J. Haematol. 2016, 175, 860. (50) Song, E.; Lee, S. K.; Wang, J.; Ince, N.; Ouyang, N.; Min, J.; Chen, J.; Shankar, P.; Lieberman, J. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 2003, 9, 347−351. (51) DeVincenzo, J.; Cehelsky, J. E.; Alvarez, R.; Elbashir, S.; Harborth, J.; Toudjarska, I.; Nechev, L.; Murugaiah, V.; Van Vliet, A.; Vaishnaw, A. K.; Meyers, R. Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res. 2008, 77, 225−231. (52) Christie, R. J.; Matsumoto, Y.; Miyata, K.; Nomoto, T.; Fukushima, S.; Osada, K.; Halnaut, J.; Pittella, F.; Kim, H. J.; Nishiyama, N.; Kataoka, K. Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection. ACS Nano 2012, 6, 5174−5189. (53) Hauser, P. V.; Pippin, J. W.; Kaiser, C.; Krofft, R. D.; Brinkkoetter, P. T.; Hudkins, K. L.; Kerjaschki, D.; Reiser, J.; Alpers, C. E.; Shankland, S. J. Novel siRNA delivery system to target podocytes in vivo. PLoS One 2010, 5, e9463. (54) Choi, Y. S.; Lee, J. Y.; Suh, J. S.; Kwon, Y. M.; Lee, S. J.; Chung, J. K.; Lee, D. S.; Yang, V. C.; Chung, C. P.; Park, Y. J. The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 2009, 31, 1429−43. (55) Kumar, P.; Ban, H. S.; Kim, S. S.; Wu, H.; Pearson, T.; Greiner, D. L.; Laouar, A.; Yao, J.; Haridas, V.; Habiro, K.; Yang, Y. G.; Jeong, J. H.; Lee, K. Y.; Kim, Y. H.; Kim, S. W.; Peipp, M.; Fey, G. H.; 1349


Review

Molecular Pharmaceutics Manjunath, N.; Shultz, L. D.; Lee, S. K.; Shankar, P. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008, 134, 577−86. (56) Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.; Loffler, K.; Fechtner, M.; Arnold, W.; Giese, K.; Klippel, A.; Kaufmann, J. A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium. Gene Ther. 2006, 13, 1222−1234. (57) Landen, C. N., Jr; Chavez-Reyes, A.; Bucana, C.; Schmandt, R.; Deavers, M. T.; Lopez-Berestein, G.; Sood, A. K. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res. 2005, 65, 6910−6918. (58) Navarro, G.; Sawant, R. R.; Biswas, S.; Essex, S.; Tros de Ilarduya, C.; Torchilin, V. P. P-glycoprotein silencing with siRNA delivered by DOPE-modified PEI overcomes doxorubicin resistance in breast cancer cells. Nanomedicine (London, U. K.) 2012, 7, 65−78. (59) Sun, T. M.; Du, J. Z.; Yan, L. F.; Mao, H. Q.; Wang, J. Selfassembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery. Biomaterials 2008, 29, 4348−4355. (60) Navarro, G.; Sawant, R. R.; Essex, S.; Tros de Ilarduya, C.; Torchilin, V. P. Phospholipid-polyethylenimine conjugate-based micelle-like nanoparticles for siRNA delivery. Drug Delivery Transl. Res. 2011, 1, 25−33. (61) Urban-Klein, B.; Werth, S.; Abuharbeid, S.; Czubayko, F.; Aigner, A. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 2005, 12, 461−466. (62) Philipp, A.; Zhao, X.; Tarcha, P.; Wagner, E.; Zintchenko, A. Hydrophobically modified oligoethylenimines as highly efficient transfection agents for siRNA delivery. Bioconjugate Chem. 2009, 20, 2055−2061. (63) Fraley, R.; Subramani, S.; Berg, P.; Papahadjopoulos, D. Introduction of liposome-encapsulated SV40 DNA into cells. J. Biol. Chem. 1980, 255, 10431−10435. (64) Schaefer-Ridder, M.; Wang, Y.; Hofschneider, P. H. Liposomes as gene carriers: efficient transformation of mouse L cells by thymidine kinase gene. Science 1982, 215, 166−168. (65) Zhang, Y.; Zhang, Y. F.; Bryant, J.; Charles, A.; Boado, R. J.; Pardridge, W. M. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin. Cancer Res. 2004, 10, 3667−3677. (66) Gao, J.; Yu, Y.; Zhang, Y.; Song, J.; Chen, H.; Li, W.; Qian, W.; Deng, L.; Kou, G.; Chen, J.; Guo, Y. EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma. Biomaterials 2012, 33, 270−282. (67) Chen, Y.; Zhu, X.; Zhang, X.; Liu, B.; Huang, L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol. Ther. 2010, 18, 1650−1656. (68) Davis, M. E.; Zuckerman, J. E.; Choi, C. H.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464, 1067−1070. (69) Hu-Lieskovan, S.; Heidel, J. D.; Bartlett, D. W.; Davis, M. E.; Triche, T. J. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res. 2005, 65, 8984−8992. (70) Yao, Y. D.; Sun, T. M.; Huang, S. Y.; Dou, S.; Lin, L.; Chen, J. N.; Ruan, J. B.; Mao, C. Q.; Yu, F. Y.; Zeng, M. S.; Zang, J. Y.; Liu, Q.; Su, F. X.; Zhang, P.; Lieberman, J.; Wang, J.; Song, E. Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis. Sci. Transl. Med. 2012, 4, 130ra48. (71) Peer, D.; Zhu, P.; Carman, C. V.; Lieberman, J.; Shimaoka, M. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 4095−4100. (72) Graham, F. L.; van der Eb, A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973, 52, 456−467.

(73) Rezaee, M.; Oskuee, R. K.; Nassirli, H.; Malaekeh-Nikouei, B. Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems. J. Controlled Release 2016, 236, 1−14. (74) Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J. Controlled Release 2011, 151, 220−228. (75) Veronese, F. M.; Pasut, G. PEGylation, successful approach to drug delivery. Drug Discovery Today 2005, 10, 1451−1458. (76) Chono, S.; Li, S. D.; Conwell, C. C.; Huang, L. An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor. J. Controlled Release 2008, 131, 64−69. (77) Yang, C.; Wei, W. The miRNA expression profile of the uveal melanoma. Sci. China: Life Sci. 2011, 54, 351−358. (78) Gao, J.; Sun, J.; Li, H.; Liu, W.; Zhang, Y.; Li, B.; Qian, W.; Wang, H.; Chen, J.; Guo, Y. Lyophilized HER2-specific PEGylated immunoliposomes for active siRNA gene silencing. Biomaterials 2010, 31, 2655−2664. (79) Gao, J.; Liu, W.; Xia, Y.; Li, W.; Sun, J.; Chen, H.; Li, B.; Zhang, D.; Qian, W.; Meng, Y.; Deng, L.; Wang, H.; Chen, J.; Guo, Y. The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes. Biomaterials 2011, 32, 3459−3470. (80) Deng, L.; Zhang, Y.; Ma, L.; Jing, X.; Ke, X.; Lian, J.; Zhao, Q.; Yan, B.; Zhang, J.; Yao, J.; Chen, J. Comparison of anti-EGFR-Fab’ conjugated immunoliposomes modified with two different conjugation linkers for siRNa delivery in SMMC-7721 cells. Int. J. Nanomed. 2013, 8, 3271−3283. (81) Khantasup, K.; Kopermsub, P.; Chaichoun, K.; Dharakul, T. Targeted small interfering RNA-immunoliposomes as a promising therapeutic agent against highly pathogenic Avian Influenza A (H5N1) virus infection. Antimicrob. Agents Chemother. 2014, 58, 2816−2824. (82) Heidel, J. D.; Yu, Z.; Liu, J. Y.; Rele, S. M.; Liang, Y.; Zeidan, R. K.; Kornbrust, D. J.; Davis, M. E. Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 5715−5721. (83) Zuckerman, J. E.; Gritli, I.; Tolcher, A.; Heidel, J. D.; Lim, D.; Morgan, R.; Chmielowski, B.; Ribas, A.; Davis, M. E.; Yen, Y. Correlating animal and human phase Ia/Ib clinical data with CALAA01, a targeted, polymer-based nanoparticle containing siRNA. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 11449−11454. (84) Hansen, B.; Linde, S.; Kolendorf, K.; Jensen, F. Absorption of protamine-insulin in diabetic patients. I. Preparation and characterization of protamine-125I-insulin. Horm. Metab. Res. 1979, 11, 85−90. (85) Liu, B. Exploring cell type-specific internalizing antibodies for targeted delivery of siRNA. Briefings Funct. Genomics Proteomics 2007, 6, 112−9. (86) He, H.; Ye, J.; Liu, E.; Liang, Q.; Liu, Q.; Yang, V. C. Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide. J. Controlled Release 2014, 193, 63−73. (87) Ayene, I. S.; Ford, L. P.; Koch, C. J. Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation. Mol. Cancer Ther. 2005, 4, 529−536. (88) Spicer, C. D.; Davis, B. G. Selective chemical protein modification. Nat. Commun. 2014, 5, 4740. (89) Bernardes, G. J.; Steiner, M.; Hartmann, I.; Neri, D.; Casi, G. Site-specific chemical modification of antibody fragments using traceless cleavable linkers. Nat. Protoc. 2013, 8, 2079−2089. (90) Liu, H.; May, K. Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. MAbs 2012, 4, 17−23. (91) Xia, C. F.; Boado, R. J.; Pardridge, W. M. Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology. Mol. Pharmaceutics 2009, 6, 747−751. (92) Sugo, T.; Terada, M.; Oikawa, T.; Miyata, K.; Nishimura, S.; Kenjo, E.; Ogasawara-Shimizu, M.; Makita, Y.; Imaichi, S.; Murata, S.; Otake, K.; Kikuchi, K.; Teratani, M.; Masuda, Y.; Kamei, T.; 1350


Review

Molecular Pharmaceutics Takagahara, S.; Ikeda, S.; Ohtaki, T.; Matsumoto, H. Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles. J. Controlled Release 2016, 237, 1−13. (93) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (94) Kim, T. I.; Kim, S. W. Bioreducible polymers for gene delivery. React. Funct. Polym. 2011, 71, 344−349. (95) Ou, M.; Xu, R.; Kim, S. H.; Bull, D. A.; Kim, S. W. A family of bioreducible poly(disulfide amine)s for gene delivery. Biomaterials 2009, 30, 5804−5814. (96) Schmid, J. A. The acidic environment in endocytic compartments. Biochem. J. 1994, 303 (Pt 2), 679−680. (97) Wyman, T. B.; Nicol, F.; Zelphati, O.; Scaria, P. V.; Plank, C.; Szoka, F. C., Jr Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997, 36, 3008−3017. (98) Lee, H.; Jeong, J. H.; Park, T. G. A new gene delivery formulation of polyethylenimine/DNA complexes coated with PEG conjugated fusogenic peptide. J. Controlled Release 2001, 76, 183−192. (99) Min, S. H.; Lee, D. C.; Lim, M. J.; Park, H. S.; Kim, D. M.; Cho, C. W.; Yoon, D. Y.; Yeom, Y. I. A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA. J. Gene Med. 2006, 8, 1425−1434. (100) Parente, R. A.; Nir, S.; Szoka, F. C., Jr Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry 1990, 29, 8720−8728. (101) Sasaki, K.; Kogure, K.; Chaki, S.; Nakamura, Y.; Moriguchi, R.; Hamada, H.; Danev, R.; Nagayama, K.; Futaki, S.; Harashima, H. An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives. Anal. Bioanal. Chem. 2008, 391, 2717−2727. (102) Oliveira, S.; van Rooy, I.; Kranenburg, O.; Storm, G.; Schiffelers, R. M. Fusogenic peptides enhance endosomal escape improving siRNA-induced silencing of oncogenes. Int. J. Pharm. 2007, 331, 211−214. (103) Mastrobattista, E.; Koning, G. A.; van Bloois, L.; Filipe, A. C.; Jiskoot, W.; Storm, G. Functional characterization of an endosomedisruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins. J. Biol. Chem. 2002, 277, 27135−27143. (104) Jiang, X.; Lok, M. C.; Hennink, W. E. Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery. Bioconjugate Chem. 2007, 18, 2077−2084. (105) Carr, J. A.; Silverman, N. The heparin-protamine interaction. A review. J. Cardiovasc. Surg. (Torino) 1999, 40, 659−666. (106) Cornetta, K.; Anderson, W. F. Protamine sulfate as an effective alternative to Polybrene in retroviral-mediated gene-transfer: implications for human gene therapy. J. Virol. Methods 1989, 23, 187−194. (107) Sorgi, F. L.; Bhattacharya, S.; Huang, L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. 1997, 4, 961−968. (108) Tabrizi, M.; Bornstein, G. G.; Suria, H. Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J. 2010, 12, 33−43.

1351


Immunoprotein-Mediated siRNA Delivery - ACS Publications

Recommend Documents